This invention relates to bacterial diseases of humans and other mammals. In particular, the invention provides a novel signaling factor involved in regulating bacterial growth and pathogenesis, analogs and derivatives of the signaling factor, and methods for controlling bacterial growth and pathogenesis through use of such analogs and derivatives.
Intercellular cooperation confers a considerable advantage on multicellular organisms that was thought to be unavailable to unicellular organisms such as prokaryotes. Research in the last twenty years has revealed, however, that prokaryotes can communicate with each other in a way that modulates gene expression, and thereby can reap benefits that would otherwise be exclusive to eukaryotes. This ability was discovered in luminous marine bacteria such as Vibrio fischeri and Vibrio harveyi, which activate the expression of genes involved in light production only when their population density exceeds a critical value. This phenomenon, known as quorum-sensing, is now recognized as a general mechanism for gene regulation in many Gram-negative bacteria, and it allows them to perform in unison such activities as bioluminescence, swarming, biofilm formation, production of proteolytic enzymes, synthesis of antibiotics, development of genetic competence, plasmid conjugal transfer, and spoliation.
Quorum-sensing bacteria fall into two classes, depending on how many density-sensing systems they have. Both classes synthesize, release, and respond to signaling molecules called autoinducers to control gene expression as a function of cell density. Bacteria in the larger class use acyl-homoserine lactone signals in a single density-sensing system, with one gene that encodes an autoinducer synthase, and another that encodes a transcriptional activator protein that mediates response to the autoinducer. These genes are homologous to luxI and luxR of V. fischeri, respectively (Bassler and Silverman, in Two component Signal Transduction, Hoch et al., eds, Am. Soc. Microbiol. Washington D.C., pp 431-435, 1995).
Many bacteria that use the autoinducer-1 signaling factor associate with higher organisms, i.e., plants and animals, at some point during their lifecycles. For example, Pseudomonas aeruginosa, an opportunistic pathogen in humans with cystic fibrosis, regulates various virulence determinants with autoinducer-1. Other examples of autoinducer-1-producing bacteria include Erwinia carotovora, Pseudomonas aureofaciens, Yersinia enterocolitica, Vibrio harveyi, and Agrobacterium tumefaciens. E. carotovora infects certain plants and creates enzymes that degrade the plant""s cell walls, resulting in what is called xe2x80x9csoft rot disease.xe2x80x9d Yersinia enterocolitica causes gastrointestinal disease in humans and reportedly produces an autoinducer. P. aureofaciens synthesizes antibiotics under autoinducer control that block fungus growth in the roots.
Bacteria of the other class, exemplified by V. harveyi, have not one but two independent density-sensing systems. V. harveyi apparently uses the more species-specific Signaling System 1 for intra-species communication, and the less species-selective Signaling System 2 for inter-species communication (Bassler et al., J. Bacteriol. 179: 4043-4045, 1997). Each system comprises a sensor-autoinducer pair; Signaling System 1 uses Sensor 1 and autoinducer-1 (AI-1), while Signaling System 2 uses Sensor 2 and autoinducer-2 (AI-2) (Bassler et al., Mol. Microbiol. 13: 273-286, 1994). While autoinducer-1 is N-(3-hydroxy butanoyl)-L-homoserine lactone (HSL) (see Bassler et al., Mol. Microbiol. 9: 773-786, 1993), the structure of autoinducer-2 has not been established, nor have the gene(s) involved in its biosynthesis been identified.
Recent research indicates that quorum-sensing takes place not only among luminous marine bacteria, but also among pathogenic bacteria, where it regulates the production of virulence factors that are critical factors in bacterial pathogenesis. Thus, it would be an advance in the art to identify and characterize compounds with autoinducer-2 activity and the genes encoding the proteins required for production of the naturally-occurring autoinducer-2. Such an advance would provide a way to identify compounds useful for controlling pathogenic bacteria, a way to augment traditional antibiotic treatments, and a new target for the development of new antimicrobial agents.
The applicants have now discovered that many bacteria, including some non-luminous pathogens, secrete a signaling molecule that mimics V. harveyi autoinducer-2 in its function and physical properties. Bacteria that produce the AI-2 signaling factor of the invention include Vibrio harveyi, Vibrio cholerae, Vibrio parahaemolyticus, Vibrio alginolyticus, Pseudomonas phosphoreum, Yersinia enterocolitica, Escherichia coli, Salmonella typhimurium, Haemophilus influenzae, Helicobacter pylori, Bacillus subtilis, Borrelia burgfdorferi, Neisseria meningitidis, Neisseria gonorrhoeae, Yersinia pestis, Campylobacter jejuni, Deinococcus radiodurans, Mycobacterium tuberculosis, Enterococcus faecalis, Streptococcus pneumoniae, Streptococcus pyogenes and Staphylococcus aureus. 
Free-living bacteria produce this novel signaling molecule only upon shifting to a colonizing, and therefore potentially pathogenic, existence in a host organism. Thus, in addition to stimulating luminescence genes in V. harveyi, the signaling molecule is expected to stimulate genes related to pathogenesis in bacteria that produce it. In addition to a purified molecule with autoinducer-2 signaling activity, the invention provides a synthetic form of the molecule and derivatives of it that regulate bacterial growth and pathogenesis.
In another aspect, there is provided a method for regulating the activity of an autoinducer-2 receptor comprising contacting an autoinducer-2 receptor with an AI-2 agonist or antagonist compound.
In another aspect, the invention provides a method of regulating autoinducer-2 activity by contacting a bacterial cell comprising autoinducer-2, or extract thereof, with a compound of structure I, II, III or IV.
In yet another aspect, the invention provides a method for regulating autoinducer-2 receptor activity by contacting an autoinducer-2 receptor with a compound of structure I, II, III or IV.
In another aspect, the invention provides a method for controlling bacterial growth or virulence by identifying a subject infected with an autoinducer-2-producing bacterium and administering to the subject a compound of structure I, II, III or IV.
In yet another aspect, the invention provides a method for inhibiting bacterial growth or virulence in a subject, by identifying a subject in which bacteria are producing autoinducer-2 and administering to the subject an inhibitor of an autoinducer-2 of the present invention.
In another aspect, the invention provides a method for identifying a compound that regulates the activity of autoinducer-2 by comparing the activity of autoinducer-2 obtained in the presence of the compound to that obtained in its absence.
In another aspect, the invention provides a method for identifying an autoinducer analog by contacting a cell, or cell extract, that produces a detectable amount of light in response to an autoinducer with the autoinducer analog and comparing the amount of light produced in the presence and the absence of the autoinducer analog.
In another aspect, the invention provides a method for identifying a compound that regulates the production or activity of autoinducer-2 by contacting with the compound a cell that produces autoinducer-2, and determining whether autoinducer-2 activity is present in the cell.
In another aspect, the invention provides a method for identifying a compound that affects binding of autoinducer-2 to an autoinducer-2 receptor by: (a) contacting autoinducer-2 and the autoinducer-2 receptor with the compound; (b) contacting (a) with a cell, or cell extract, that produces light in response to autoinducer-2 binding to the autoinducer-2 receptor; and (c) measuring the effect of the compound on light production.
In yet another aspect, the invention provides a method for identifying a compound that affects autoinducer-2 binding to an autoinducer-2 receptor by: (a) contacting with the compound a complex formed between autoinducer-2 and the autoinducer-2 receptor to allow dissociation of the complex; (b) contacting (a) with a cell, or cell extract thereof, that produces light in response to binding of autoinducer-2 to the autoinducer-2 receptor; and (c) measuring the effect of the compound on light production.
In yet another aspect, the invention provides a method for regulating expression of a siderophore in a bacterial cell by contacting a cell capable of producing the siderophore with a compound of structure I, II, III or IV.
In another aspect, the invention provides a method for regulating exopolysaccharide production in a cell by contacting a cell capable of producing an exopolysaccharide with a compound of structure I, II, III or IV.
In another aspect, the invention provides a method for regulating bacterial colony morphology by contacting a bacterial colony with a compound of structure I, II, III or IV.
In another aspect, the invention provides a method for regulating bacterial biofilm formation by contacting a bacterium capable of biofilm formation with any combination of compounds set forth in structures I-IV.
In another aspect, the invention provides a method for producing autoinducer-2 by contacting S-adenosylhomocysteine (SAH) with a LuxS protein.
In another aspect, the invention further provides a method for producing autoinducer-2 by; a) contacting S-adenosylhomocysteine (SAH) with a 5xe2x80x2-methylthioadenosine/S-adenosylhomocysteine nucleosidase (pfs) protein to form S-ribosylhomocysteine; b) contacting the S-ribosylhomocysteine from a) with a LuxS protein to promote the conversion of S-ribosylhomocysteine to autoinducer-2.
In yet another aspect, the invention further provides a method for detecting an autoinducer-associated biomarker by: (a) contacting at least one cell with an autoinducer to promote induction of a biomarker; and (b) detecting the biomarker.
In another aspect, the invention provides a method for regulating bacterial cell growth or expression of a virulence factor comprising contacting a bacterial cell with an isolated autoinducer-2 analog comprising the structure: 
The invention further provides a synergistic antibiotic composition comprising an antibiotic and an inhibitor of the quorum-sensing pathway of a microorganism.
The invention further provides a medical device comprising a synergistic antibiotic composition comprising an antibiotic and an inhibitor of the quorum-sensing pathway of a microorganism, as well as a method of treating infections in a warm-blooded animal caused by microorganisms possessing a quorum-sensing mechanism, comprising administering to the animal a therapeutically effective amount of the synergistic antibiotic composition.
The invention further provides a pharmaceutical composition comprising a synergistic antibiotic composition comprising an antibiotic and an inhibitor of a quorum-sensing pathway of a microorganism or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable carriers, adjuvants or vehicles.
The invention also provides a method of treating infections in a warm-blooded animal caused by microorganisms possessing a quorum-sensing mechanism that comprises administering to the animal a therapeutically effective amount of the synergistic antibiotic composition comprising an antibiotic and an inhibitor of the quorum-sensing pathway of a microorganism.
The invention further provides a method for inhibiting biofilm formation by contacting a bacterium capable of biofilm formation with a compound having the structure set forth in structure III or structure IV.
The invention further provides a medical device comprising at least one antimicrobial compound having the structure set forth in structure III or structure IV, where the device is supplemented with the compound and the compound is present in a concentration sufficient to provide a localized antimicrobial effect.
The invention further provides a medical device comprising at least one synergistic antibiotic composition of an antibiotic and an inhibitor of the quorum-sensing pathway of a microorganism, where the composition is present in a concentration sufficient to provide a localized antimicrobial effect.
The invention further provides a medical device comprising at least one pharmaceutical composition comprising a synergistic antibiotic composition comprising an antibiotic and an inhibitor of a quorum-sensing pathway of a microorganism or a pharmaceutically-acceptable salt thereof and one or more pharmaceutically acceptable carriers, adjuvants or vehicles, wherein the composition is present in a concentration sufficient to provide a localized anti-microbial effect.
Additional features and advantages of the present invention will be better understood by reference to the drawings, detailed description and examples that follow.